We studied the ionization process of aromatic carboxylic acids, including ones with or without hydroxy groups in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), because many natural products, metabolites, and drags contain those structural units. In the actual experimental data, benzoic acid was ionized as only deprotonated molecule [M－H]－. In contrast, both of negative molecular ion M－ and deprotonated molecule [M－H]－ were generated from 2-naphthoic acid and 2-anthracenecarboxylic acid, and the ratio of negative molecular ion to deprotonated molecule M－/[M－H]－ was increased in 2-anthracenecarboxylic acid. In addition, the ratio of 2-anthracenecarboxylic acid was much higher than those of 1- and 9-anthracenecarboxylic acids among the three isomers. Therefore, 2-substitution induced the generation of the negative molecular ion M－, which can made us prediction of the substituted positions from their overlapping peak isotope patterns. 2,5-Dihydroxybenzoic acid showed two deprotonated molecules, [M－H]－ and [M－H*－H]－, which was generated from a neutral hydrogen radical (H*) removal from a phenolic hydroxy group. The deprotonated molecule [M－H*－H]－ of 2,5-DHBA was the most abundant among six dihydroxybenzoic acids and three hydroxybenzoic acid. This observation raises the possibility that such a property of 2,5-DHBA could be a clue to explain its highest efficiency as a MALDI matrix. The order of the hydrogen radical removal from the phenolic hydroxy groups was the 3-<4-<<5-positions in the dihydroxybenzoic acids, and the 3-<4-positions in hydroxybenzoic acids. The intra-molecular hydrogen bonding between 1-carboxy and 2-hydroxy groups was an important factor in hydrogen radical removal in the hydroxylbenzoic acids and dihydroxybenzoic acids.